The vertebrate immune system established different ways to detect invading pathogens based on certain characteristics of their microbial nucleic acids. Detection of microbial nucleic acids alerts the immune system to mount the appropriate type of immune response that is required for the defense against the respective type of pathogen detected. Detection of viral nucleic acids leads to the production of type I interferon (IFN) including IFN-α and IFN-β, the key cytokines for anti-viral defense.
IFN-α was the first type of interferon to be identified and commercialized; it is widely used clinically in the treatment of a variety of tumors (e.g., hairy cell leukemia, cutaneous T cell leukemia, chronic myeloid leukemia, non-Hodgkin's lymphoma, AIDS-related Kaposi's sarcoma, malignant melanoma, multiple myeloma, renal cell carcinoma, bladder cell carcinoma, colon carcinoma, cervical dysplasia) and viral diseases (e.g., chronic hepatitis B, chronic hepatitis C). IFN-α products that are currently in clinical use include the recombinant protein and the highly purified natural protein, both of which have high production costs. Therefore, there is a need for more economical ways of providing IFN-α to patients in need. Furthermore, IFN-α is currently administrated systematically and causes a broad spectrum of side effects (e.g. fatigue, flu-like symptoms, diarrhea). Most alarmingly, IFN-α causes a decrease in bone marrow function which leads to increased susceptibility to life-threatening infections, anemia and bleeding problems. Therefore, there is a need for ways of providing IFN-α in a more localized (i.e., target-specific) matter to reduce the occurrence of side effects.
Receptor-mediated detection of pathogen-derived nucleic acids assists in protecting the host genome from invading foreign genetic material. A new picture is evolving in which the ability of biological systems to detect viral nucleic acids via protein receptor-nucleic acid ligand interactions is crucial for maintaining the integrity of the genome and for survival.
A number of receptor proteins have evolved that take part in nucleic acid recognition. Recent studies indicate that one of the most important protein receptors for antiviral defense is the retinoic-acid-inducible protein I (RIG-I), a member of the helicase family containing two caspase-recruitment domains (CARDs) and a DExD/H-box helicase domain (M. Yoneyama et al., Nat Immunol 5, 730 (July, 2004)). RIG-I-mediated recognition of a specific set of RNA viruses (flaviviridae, paramyxoviridae, orthomyxoviridae and rhabdoviridae) (M. Yoneyama et al., Nat Immunol 5, 730 (July, 2004); R. Sumpter, Jr. et al., J Virol 79, 2689 (March, 2005); H. Kato et al., Nature 441, 101 (Apr. 9, 2006)) has a critical role in antiviral host defense in vitro and in vivo. A second member of the helicase family, MDA-5, is responsible for the antiviral defense against a reciprocal set of RNA viruses (picornaviridae)(H. Kato et al., Nature 441(7089):101-105, Apr. 9, 2006).
In addition to RIG-I and MDA-5, the four members of the Toll-like receptor (TLR) family, TLR3, TLR7, TLR9 and TLR9, are also known to be involved in viral nucleic acid recognition. RIG-I and MDA-5 differ from the TLRs in their subcellular localization, expression pattern, signal transduction pathways and ligands.
While RIG-I and MDA-5 are cytosolic receptors, TLR3, TLR7, TLR8 and TLR9 are located in the endosomal membrane.
While TLRs are mainly expressed on certain defined immune cell subsets (i.e. TLR9 restricted to PDC and B cells), RIG-I and MDA-5 are expressed in both immune and non-immune cells (H. Kato et al., Immunity 23, 19 (July, 2005)).
Besides distinct expression profiles and cellular localization, signalling of endosomal TLRs and the two cytoplasmic receptors RIG-I and MDA-5 differs. While TLR3 signals via TRIF and TLR7, TLR8 and TLR9 signal via MyD88, RIG-I recruits a CARD-containing adaptor, IPS-1 (T. Kawai et al., Nat Immunol 6, 981 (October, 2005)) (also known as MAVS (R. B. Seth et al., Cell 122, 669 (Sep. 9, 2005)), VISA (L. G. Xu et al., Mol Cell 19, 727 (Sep. 16, 2005)) or Cardif (E. Meylan et al., Nature 437, 1167 (Oct. 20, 2005))). IPS-1 relays the signal to the kinases TBK1 and IKK-i, which phosphorylate interferon-regulatory factor-3 (IRF-3) and IRF-7, transcription factors essential for the expression of type-I interferons. As a consequence, in vivo, endosomal and cytoplasmic nucleic acid receptors induce different cytokine patterns. For example, both TLR3 and MDA-5 contribute to IL-12 production in response to poly(I:C), while MDA-5 but not TLR3 is responsible for IFN-α induction (H. Kato et al., Nature 441, 101 (Apr. 9, 2006)).
The ligand for TLR3 is long dsRNA such as poly(I:C) (L. Alexopoulou, et al., Nature 413, 732 (Oct. 18, 2001)), for TLR7 ssRNA (S. S. Diebold et al., Science 303, 1529 (Mar. 5, 2004); F. Heil et al., Science 303, 1526 (Mar. 5, 2004)) and short dsRNA with certain sequence motifs (i.e., the immunostimulatory RNA, isRNA) (V. Hornung et al., Nat Med 11, 263 (March, 2005)), and for TLR9 CpG DNA (A. M. Krieg et al., Nature 374, 546 (Apr. 6, 1995); H. Hemmi et al., Nature 408, 740 (Dec. 7, 2000)).
In several studies, long double-stranded RNA was proposed to be the ligand for MDA-5 and RIG-I (M. Yoneyama et al., Nat Immunol 5, 730 (July, 2004); H. Kato et al., Nature 441, 101 (Apr. 9, 2006); S. Rothenfusser et al., J Immunol 175, 5260 (Oct. 15, 2005)). A synthetic mimic of long dsRNA is poly(I:C). Recent data showed that poly(I:C) is a ligand for MDA-5, while it is not recognized by RIG-I (H. Kato et al., Nature 441, 101 (Apr. 9, 2006)). On the other hand, long dsRNA was found to activate RIG-I but not MDA-5 (H. Kato et al., Nature 441, 101 (Apr. 9, 2006)). This discrepancy of long dsRNA and poly(I:C) activity suggests that there is more to cytoplasmic RNA recognition than long dsRNA.
In general, compartmentalization and different molecular structure are believed to contribute to the detection of foreign nucleic acids. DNA (G. M. Barton et al., Nat Immunol 7, 49 (January, 2006)) and RNA (F. Heil et al., Science 303, 1526 (Mar. 5, 2004)) localized in the endosome or DNA localized in the cytoplasm (K. J. Ishii et al., Nat Immunol 7, 40 (January, 2006)) are recognized and thus interpreted as foreign. The frequency of so-called CpG motifs in microbial DNA serves as a molecular feature further improving distinction of self and non-self DNA in the endosome. Although RNA recognition in the endosome is sequence dependent (F. Heil et al., Science 303, 1526 (Mar. 5, 2004); V. Hornung et al., Nat Med 11, 263 (March, 2005)), no sequence motifs have been defined so far that serve as a molecular basis to improve distinction of self and non-self RNA (i.e. motifs that are more frequent in viral than in self RNA) in the cytoplasm. Instead, the molecular characteristic of double-strandedness seems to allow distinction of self and non-self RNA. In fact, in the endosome, long double-stranded RNA and its mimic poly(I:C), but not single-stranded RNA, are recognized via TLR3 (L. Alexopoulou, et al., Nature 413, 732 (Oct. 18, 2001)). In the cytoplasm, abundant self RNA complicates our understanding of the recognition of non-self RNA. Nevertheless, the concept that long dsRNA in the cytoplasm is detected as non-self has never been questioned since the discovery of type I IFN.
Unlike in the absence of RIG-I and MDA-5, antiviral defense is largely maintained in the absence of TLRs (A. Krug et al., Immunity 21, 107 (July, 2004); K. Tabeta et al., Proc Natl Acad Sci USA 101, 3516 (Mar. 9, 2004); T. Delale et al., J Immunol 175, 6723 (Nov. 15, 2005); K. Yang et al., Immunity 23, 465 (November, 2005)), underscoring the critical role of RIG-I and MDA-5 in antiviral responses.
It is therefore an object of the present invention to provide polynucleotides/oligonucleotides which are capable of stimulating an anti-viral response, in particular, a type I IFN response. It is another object of the present invention to provide a pharmaceutical composition capable of inducing an anti-viral response, in particular, type I IFN production, in a patient for the prevention and treatment of diseases and disorders such as viral infection. It is also an object of the present invention to provide a pharmaceutical composition for treating tumor.
A recent study demonstrated that in vitro transcribed siRNAs (small-interfering RNA), but not synthetic siRNAs, stimulated the production of type I IFN from selected cell lines (D. H. Kim et al., Nat Biotechnol 22, 321 (March, 2004); US 2006/0178334). However, the structural requirements and the physiological relevance of this induction and the mechanism of detection remain unclear. Furthermore, in the work by Kim et al., the in vitro transcribed siRNAs, regardless of their nucleotide sequence, induced type I IFN production in both virally infected and non-infected cells, regardless of whether the target mRNAs were present or not, leading to cell death. In other words, the in vitro transcribed siRNAs induced IFN production and consequently, cell death, in a non-sequence-dependent and non-target cell-specific manner. The lack of sequence- and cell-specificity severely limits, if not precludes, the use of such in vitro transcribed siRNAs for therapeutic purposes.
It is therefore a further object of the present invention to provide polynucleotides/oligonucleotides which are capable of inducing an anti-viral response, in particular, a type I IFN response, in a nucleotide sequence-dependent and target cell-specific manner. Such polynucleotides/oligonucleotides can be advantageously used for the treatment of diseases and disorders such as viral infection and tumor without harming bystander (i.e., healthy, non-infected or non-diseased) cells.